KR20020010327A - Expression vector using for animal cell - Google Patents

Abstract

PURPOSE: An expression vector capable of expressing foreign proteins in animal cells is provided, therefore the foreign proteins can be effectively expressed in animal cells with maintaining the structure and function of foreign protein. CONSTITUTION: The expression vector capable of expressing foreign proteins in animal cells comprises MAR(Nuclear matrix attachment region) element of a beta-globin gene or reverse oriented MAR element of a beta-globin gene in its 5'-terminal, in which the expression vector is pMS (KCCM 10203) having the nucleotide sequence of SEQ ID NO: 1. The expression vector capable of expressing foreign proteins in animal cells comprises a terminal sequence combined with poly-A(polyadenylation) of SV40 virus having the nucleotide sequence of SEQ ID NO: 3 and a terminal sequence of gastrin gene, in which the expression vector is pSG having the nucleotide sequence of SEQ ID NO: 7.

Description

Expression vector using for animal cell}

The present invention relates to an animal cell expression vector, and more particularly, an expression including a transcription termination site of a beta-globin gene MAR (Nuclear Matrix Attachment Region; hereinafter referred to as MAR) and / or a gastrin gene. It's about vectors.

Currently, a variety of expression systems, such as microorganisms, plants, yeasts, insect cells, animal cells, etc. are used to express and obtain a large amount of a target protein and to use it for treatment. The most easily used expression system is a microbial expression system and various microbial expression systems have been developed and commercialized.

However, microbial expression systems have some limitations. The biggest limiting factor is the expression of microorganisms and the mechanism of protein modification (glycosylation, phosphorylation, and amidation). Even though animal cells express the same genes in microbial systems, the structure and characteristics of the expressed protein are different. It is not exactly the same as the protein in the cell. Therefore, the recombinant protein using the microbial expression system is currently limited to proteins that have little functional change, even if the protein is hardly modified or the protein is slightly modified or modified. In addition, in using a recombinant protein using a microbial expression system, there is a hassle that must be accompanied by a secondary contaminant removal process due to microbial contamination, microbial endotoxin contamination.

In contrast, although animal cell expression systems are the most suitable systems for expressing animal proteins, the expression efficiency of recombinant proteins is lower than that of microbial expression systems, resulting in high production costs and difficulty in manipulating animal cells. have.

Currently used animal cells include Chinese Hamster Ovary (CHO), Baby Hamster Kidney (BHK), myeloma (myeloma), etc., and the expression vector of the foreign protein is introduced by introducing an expression vector into the cell line in the same manner as the microbial expression system. .

However, in general, since the expression level of foreign genes is very small, various methods of genetic manipulation are performed. In the case of CHO cell lines, methotrexate, which is an inhibitor of DHFR (Dihydrofolate reductase) enzyme, is added to the cell culture medium. A method of securing a highly expressed CHO cell line which selectively survives MTX and increases the number of genes and thus the expression of proteins is used.

In general, when inducing foreign gene expression in animal cells, foreign genes are co-introduced with a vector having a marker, and the transformed cells are selected through a culturing process in a selective medium over a long time. However, as mentioned above, their expression frequency is very low in most cases. One of the reasons for the low frequency of foreign protein expression is that, unlike microbial systems, these foreign genes are inserted into chromosomes in host cells. In addition, it is difficult to predict the expression of external genes of stable transfectants in which foreign genes are stably inserted into the host cell chromosome. This is because there is no correlation between the number of exogenous genes and gene expression in animal cells (Grindley et al., 1987. Trends Genet . 3, 16-22; Kucherlapati et al., 1984. Crit. Rev. . Biochem 16, 349-381;.. .. Palmiter et al, 1986. Annu Rev. Genet 20, 465-499) Therefore, in most cases, is suppressed by the surrounding nucleotide is inserted gene expression in animal cells, stable Stably integrated transgenes but are often expressed at low levels (Eissenberg et al., 1991. Trends Genet . 7, 335-340; Palmiter et al., 1986. Annu. Rev. Genet . 20, 465-499)

The availability of nucleic acid factors has been reported in several systems to protect the expression of foreign genes from these gene location specific effects. Nucleic acid sites collectively referred to as buffering factor and nuclear matrix attachment region (hereinafter referred to as MAR) or scaffold attachment region (SAR) are attempted to target the nucleic acid factor. It became.

Kalos (Kalos et al., 1995.Mol. Cell. Biol. 15, 198-207) combine the MAR factor of the human apolipoprotein B gene with a minimal promoter transgene construct and induce gene expression by host transfection in a variety of cells. Expression of the gene transcript was increased about 200 fold. Similarly, the MAR factor of the chicken lysozyme A gene and the SAR element of the beta-interferon gene in vertebrate cells also increase the expression of foreign genes regardless of the chromosomal insertion site of the host cell. (Eissenberg et al., 1991.Trends genet. 7, 335-340; Klehr et al., 1991. Biochemistry 30, 1264-1270) However, there have not been any attempts by these MAR or SAR factors to substantially increase protein production or industrial profitability in CHO cell lines.

In addition, in the gene expression of animal cells, a process such as migration of mRNA occurs in order to form proteins from mRNA transcribed by a promoter. At this time, mRNA stability is directly related to the final protein production yield.

The gene transcription termination site included in the expression vector of the animal cell affects intracellular mRNA stability by regulating poly-A (polyadenylation) in gene expression. The transcription termination region of the gene is composed of three regions, a poly-A signal, a cleavage region, and a termination region. The adenylation signal is best studied as a site having an AATAAA sequence, whereas a polyadenylation cleavage occurs. The site and the termination site, the site where gene transcription is terminated by RNA polymerase II, are not well known. In addition, the GU / U-rich region, etc. in addition to the three regions essential for the termination of gene transcription has recently been reported as a site for regulating polyadenylation of mRNA, the detailed mechanism is not known yet.

To date, expression vectors using animal cells mainly use poly-A signals of SV40 virus and bovine growth hormone (BGH) genes, and improve the termination sites that directly affect mRNA stability. The plan is not working.

Accordingly, an object of the present invention is to provide an animal cell expression vector capable of increasing expression efficiency in expressing foreign proteins using an animal cell system.

1 illustrates the structure of a vector prepared by modifying a pSV-βgal vector to prepare an expression vector of the present invention.

Figure 2 shows the incidence of β-gal gene by various MAR and SAR factors used in the present invention,

Figure 3 illustrates the effect of various MAR and SAR factors of the present invention on β-gal expression through the protein activity of β-gal expressed in the cell line,

Figure 4 shows a mutant of the beta globin MAR factor prepared in the present invention,

Figure 5 is a measure of the expression frequency and β-gal expression of β-gal protein after introducing a mutant vector of beta globin MAR factor prepared in the present invention into CHO cells,

FIG. 6 shows the number of β-gal genes (control: a, the present invention: b) and the number of transcribed RNAs in animal cells including the control vector pSV-β-gal or pMS-β-gal vector of the present invention. c, the present invention: d), and the selectable neo gene number (control: e, the present invention: f) was analyzed by Southern blot and Northern blot,

7 is a graph (g) showing the correlation between the number of β-gal genes and the amount of β-gal expression inserted into an animal cell line including the control vector pSV-β-gal or pMS-β-gal vector of the present invention. Will,

8 shows the expression titers of pMS-β-gal vectors of the present invention in various cell lines,

Figure 9 shows the change in the expression level of the foreign protein according to the amount of MTX addition in the cells introduced pMS-β-gal vector or control vector of the present invention,

Figure 10 shows the expression activity of the scu-PA protein in the introduced cell line after insertion of the single chain proUrokinase (scu-PA) gene in the pMS vector and pMC vector of the present invention, compared with the control group,

11 shows a combination of a transcription terminator and an SV40 poly-A signal of the gastrin gene of the present invention,

12 illustrates the expression levels of β-gal in expression vectors having a combination of a transcription terminator and an SV40 poly-A signal as a transcription terminator of the present invention,

Figure 13 shows the structure of the expression vector pMSG of the present invention,

Figure 14 shows the expression pattern of TGF-βSRII of the pMSG vector of the present invention by an antigen antibody response,

15 shows the amount of TGF-βSRII expression induced by cloning the TGF-βSRII gene in the pMSG vector of the present invention, transforming the CHO cell line, and then adding MTX to TGF-βSRII expressing cells.

In order to achieve the above object, the present invention provides an animal cell expression vector comprising a reverse matrix of MAR factor (Nuclear Matrix Attachment Region) of the beta globin gene or MAR factor of the beta globin gene at the end of a conventional vector promoter 5 '. .

In another aspect, the present invention provides an animal cell expression vector comprising a combination of a transcription termination region of a gastrin gene in a poly-A (polyadenylation) signal of the SV40 virus of SEQ ID NO: 3 as a transcription terminator in a conventional vector. .

The present invention also provides a pMSG vector of SEQ ID NO: 8 comprising a beta-globin Nuclear Matrix Attachment Region element reverse of the beta globin gene and a transcription terminator combination of poly-A and gastrin genes of the SV40 virus. to provide.

In another aspect, the present invention is an animal cell expression vector comprising a reverse matrix of the MAR factor (Nuclear Matrix Attachment Region) of the beta globin gene or the MAR factor of the beta globin gene at the 5 'terminal of the conventional vector promoter, transcription termination factor to the conventional vector By using a vector selected from the group consisting of an animal cell expression vector and a pMSG expression vector comprising a combination of the transcription termination site of the gastrin gene in the poly-A (polyadenylation) signal of the SV40 virus of SEQ ID NO: 3 It provides a method for producing a bioactive substance in cells.

Hereinafter, the present invention will be described in detail.

The inventors have devised an optimal expression vector for the problem of position-specific expression effects and the improvement of gene expression in the expression of foreign genes in an animal expression system.

Animal cell expression vector of the present invention is a further addition of the nucleotide sequence useful in the existing expression vector. This useful sequence protects the expression of foreign genes from the site-specific inhibitory effects of host cell chromosomes in CHO (Chinese hamster ovary) cells or BHK (Baby hamster kidney) cells, which are commonly used as commercial animal cells. Nuclear matrix attachment regions (MAR) and scaffold attachment regions (SAR) were used to design factors that increase expression. The MAR factor or SAR factor was added to the 5 'end of the promoter to analyze the performance of the expression vector. In order to secure the nucleotide sequence of the MAR factor, the genomic nucleic acid was purified from the cell, and then cloned into the E. coli cell vector using a genomic nucleic acid PCR method and subcloning method to obtain various MAR and SAR factors. The MAR factor and SAR factor are chicken lysozyme 5 'MAR (Phi-Van, L. and Stratling, WH, Biochemistry 35, 10735-10742 (1996), gene bank #: X98408), chicken phi alpha globin 5' MAR (Kraevskii, VA, Mikhailov, VS and Razin, SV, Mol. Biol . 26, 672-678 (1992), gene bank #: X64113), human beta globin 5 'MAR (Yu, J., Bock, JH, Slightom , JL and Villeponteau, B., Gene 139 (2), 139-145 (1994), gene bank #: L22754), CHO DHFR intron MAR (Kas, E. and Chasin, LA, J. Mol. Biol . 198 (4) ), 677-692 (1987), gene bank #: X06654), human HPRT intron MAR (Sykes, RC, Lin, D., Hwang, SJ, Framson, PE and Chinault, AC, Mol. Gen. Genet . 212, 301-309 (1988), gene bank #: X07690), human CSP-B gene flanking SAR (Hanson, RD and Ley, TJ, gene bank #: M62716), and human interferon beta gene flanking SAR ( Mielke, C., Kohwi, Y., Kohwi-Shigematsu, T. and Bode, J., Biochemistry 29, 7475-7485 (1990), gene bank #: be selected from the group consisting of M83137) bar Preferable.

The seven MAR or SAR factors secured above were inserted into the animal cell expression vector and the β-gal present in the expression vector was expressed to confirm the correlation between expression titers and MAR / SAR factors. First, a recombinant vector having a useful multicloning site (MCS) inserted into the pSV-β-gal vector promoter (5 ') by in vitro PCR mutagenesis was prepared as shown in FIG. (Version I and version II vectors) The final test vector was completed by inserting the seven MAR or SAR factors obtained above into the promoter of the prepared vector, and then DOSPER (a type of fatty surfactant in CHO DG44 cells). Boehringer Manheim (Germany) was used to introduce 2 ug of test vectors with the pSV2Neo vector, which is a selection vector, respectively, and the pSV-β-gal vector was also performed as a control. Transformed CHO DG44 cells were screened in medium containing G418 (neomycin) and then the frequency of β-gal-expressing cell lines using β-gal staining (checked by blue staining using IPTG and X-gal). The expression potency was confirmed by measuring the change in absorbance. Expression titration was carried out in two aspects, specifically, by measuring the expression frequency to identify the number of cells expressing β-gal and the amount of expression in the cell lines expressing β-gal.

Figure 2 shows the effect of various MAR and SAR factors used in the present invention on the frequency of the positive cell line expressing β-gal gene, β-gal expression in the case of pSV-β-gal vector used as a control The frequency of positive cell lines was significantly increased in the case of beta globin MAR, CSP-B SAR, and interferon beta SAR factor compared to the frequency of 20-30%, especially about 70-80% of β- beta globin MAR. The frequency of gal expression was observed.

In addition, in the novel animal cell expression vector through the introduction of the MAR factor of the present invention, in the same manner as described above to investigate the effect of the combination form of the MAR factor and the SV40 promoter on the function of the existing SV40 promoter G418 resistant cell lines were selected and the production of β-gal protein, a foreign marker gene, was investigated. As shown in FIG. 2, since the frequency of cell lines induced using each MAR factor is different, the β-gal staining method and the β-gal enzyme assay were used to compare the amount of β-gal protein expressed in the positive cell line. Simultaneously performing the method of examining the expression level of the protein was compared and analyzed the activity of β-gal protein in the same number of positive cells.

Figure 3 shows the β-gal protein activity expressed in a cell line expressing various MAR and SAR factors of the present invention, β-gal protein production amount per positive cell is pSV-β-gal control group When the beta globin MAR, CSP-B SAR, and the interferon beta SAR factor were used, the activity of β-gal, ie, the expression amount, was increased compared to the expression vector of. In the vector using beta globin MAR factor, β-gal expression increase of about 7 times or more was observed.

Therefore, the MAR factor of the present invention is more preferably human beta globin MAR factor.

In addition, in order to enhance the effect and usefulness of the beta globin MAR factor, the nucleic acid sequence of the beta globin MAR factor was analyzed. The beta globin MAR factor consists of 2,988 nucleotide sequences, and their detailed functional composition is unknown, but A + T (consensus nucleic acid sequence and A + T (in nucleic acid distribution), which are commonly found in the intermediate MAR factor, are not known. Alu factor of 244 bp exists in the 800 bp region and 3 'region, where the ratio of adenine and thymidine is high. Alu nucleic acid sites are usually 300 bp in size and are usually composed of two directly repeating monomer units. Thousands of homologs are present on chromosomes of eukaryotic cells, causing frequent recombination. (Jagadeeswaran et al., 1982. Nature 296, 469-470; Rogers. 1985. Int. Rev. Cytol . 93, 187-279) The above characteristics indicate that the alu factor in the beta globin MAR factor is SV40. When present in the front of the promoter can be a factor that inhibits normal gene expression when cells are cultured for a long time.

Thus, we have constructed a mutant of beta globin MAR factor to rule out the negative effects and improve the performance of beta globin MAR factor.

Figure 4 shows a mutant of the beta globin MAR factor of the present invention, prepared by mutants c, d, e, f, g, h, i of the reverse b and the beta globin MAR factor of beta globin MAR factor Then, the expression vector was prepared by introducing the promoter of the pSV-β-gal version I vector of FIG. 1.

After introducing the mutant vector of the beta globin MAR factor prepared above into CHO cells, the expression cell frequency and the amount of β-gal expression of β-gal protein were measured and shown in FIG. 5. Most of the mutants of the beta globin MAR factor had reduced β-gal expression titres compared to the pSV-beta globin MAR-β-gal vector, whereas the pMS-β-gal expression vector containing the reverse of beta globin MAR factor Β-gal expression titers were observed in cell lines transformed with. In addition, as shown in FIG. 6 and FIG. 7, the correlation between the number of inserted genes and the amount of expressed protein was not correlated with the number of inserted genes and the amount of expressed protein, but was expressed as pMS-β-gal. In the transformed vector, it was confirmed that a proportional relationship was established between the number of inserted genes and the amount of expressed protein. This not only reduces the likelihood of future recombination by positioning the alu factor in the opposite direction of the promoter, but also allows the beta globin MAR reverser to have a higher expression titer than the globin MAR factor and is dependent on the insertion position. Overcoming the expression inhibition phenomenon was found to increase the protein expression. Therefore, the MAR factor of the present invention was most preferably the reverse of beta globin MAR factor.

In accordance with the present invention, the MAR or SAR factor may be increased in promoter 5 'of an existing expression vector to increase expression of the foreign protein, and a mutant or reverse of MAR or SAR factor may be inserted into an existing expression vector. It was confirmed that the expression potency of the protein can be increased. Therefore, the present invention provides an expression vector comprising a mutant or a reverse of the expression vector containing the MAR factor or SAR factor and MAR factor or SAR factor. The MAR factor or SAR factor is phi-a MAR (chicken pi α-globin 5 'MAR), beta globin MAR (human β-globin 5' MAR), DHFR MAR (CHO DHFR intron MAR), HPRT MAR (human HPRT intron) Preferably selected from the group consisting of MAR), CSP-B gene flanking SAR element (CSP-B MAR), human interferon-β gene flanking SAR element (CAR), and lyso MAR (Chicken lysozyme 5 'MAR). Do.

Among them, pMS KCCM 10203 including the reverse product of the beta globin MAR factor, which is a vector prepared in the present invention, is a 6287 bp vector in which the reverse body of human beta globin MAR factor is located at 5 'of the SV40 virus promoter and has multiple cloning sites. By inserting other genes into many cloning sites, foreign genes can be expressed. The base sequence of the pMS vector was prepared as SEQ ID NO: 1 in Sequence Listing.

As a result of analyzing the expression of genes in the CHO cell line environment after constructing pMS-β-gal with β-gal inserted into the pMS vector, it was found that the beta globin MAR factor reverser was used compared with the conventional SV40 promoter. The expression frequency of 4 to 4 times and the amount of foreign gene expression of about 7 to 10 times increased. In addition, the pMS-β-gal vector system is applicable to various animal cells as shown in Figure 9, BHK cells, CHO cells, NIH 3T3 cells, and HEK 293 is preferred. In addition, as shown in FIG. 9, when inducing foreign protein expression with the pMS-β-gal vector system, MTX (methotrexate) may be added to further increase the protein expression activity.

In the present invention, the foreign protein expression titers of MAR or SAR factors were tested using CMV (cytomegalo virus) -derived promoters, which are widely used in addition to the promoter of SV40 virus. Since the titer of the gene expression promoter shows various variations depending on the type of promoter in various cell lines, in the present invention, the promoter of CMV virus was further performed in addition to the SV40 virus. Therefore, in order to investigate the effect of the beta globin MAR reverser selected in the present invention on the function of the CMV-derived promoter, a gene of scu-PA (short-chain urokinase), which is a human-derived protein, was constructed after the pMS vector, similar to the pMS vector. pMC, and control vectors were produced.

FIG. 10 illustrates the expression titers of scu-PA by introducing pMSPUK, pMCPUK, pSPUK and pMCPUK vectors prepared by introducing scu-PA into pMS, pMC, and control vectors pSV and pCMV into CHO cell lines. It was observed that the gene expression was increased by about four times more than the control vectors pSV and pCMV expression vectors. Thus, the betaglobin MAR reverser of the present invention can be used to express proteins in animal cells in appropriate combination with various promoters.

In addition, the vector further comprising the beta globin MAR reverse body of the present invention can be obtained by inserting a foreign gene useful proteins such as bioactive substances in a conventional manner in animal cell lines.

In addition, the present inventors screened and produced a gastrin gene transcription termination site of the human body in which the poly-A signal, cleavage site, and termination site, which are essential for gene transcription termination, are known and further applied to the expression vector of the present invention. In order to establish the uniqueness of the expression vector and at the same time increase the mRNA stability to improve the efficiency of the expression vector.

Gastrin consists of 603 bp fragments containing poly-A signal, cleavage site, and termination site on HindIII restriction enzyme map. The base sequence is set forth in SEQ ID NO: 2. The poly-A signal and the cleavage site are 15 bp apart and the termination site is about 220 bp away from the poly-A signal. The transcription termination site of gastrin located at the above-mentioned distance is terminated transcription at the termination site and cleavage of mRNA and poly-A at the cleavage site.

The present inventors prepared a combination capable of improving foreign gene expression titers by combining the SV40 poly-A signal and the gastrin gene transcription terminator as shown in FIG. 11 to confirm the expression titers of foreign proteins in animal cell lines.

Figure 11 shows a combination of the transcription terminator and SV40 poly-A signal of the gastrin gene, the transcription termination site of the present invention is the SPA (SV40 polyadenylation signal) combination of the pSV vector set as a control of a , pSG-GTF (gastrintermination site) combination of b, pSV-SPA-GTR (reverse of GTF) combination of c, pSV-GPA (gastrin polyadenylation signal) combination of d, pSV-GPA-GTF combination of e , pSV-SPA-GTR combination of f, g pSV-GMPA (mutant of GPA) combination, pSV-GMPA-GTF combination of h, and pSV-GMPA-GTR combination of i are preferred. Do. The base sequences of the SPA-GTF, SPA-GTR, SP-GPA, and SPA-GMPA were prepared using SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. The combinations of the groups were inserted into pSV-β-gal vectors, respectively, and then introduced into Cos-7 cell lines to measure the expression titers of β-gal. The expression titers are expressed in β-gal as shown in FIG. 12. Among the various combinations, the pSG expression vector, that is, the combination of the SV40 poly-A signal and the gastrin transcription terminator, reduced the amount of protein expression compared to the conventional SV40 poly-A signal. 4 times increase effect was obtained. Therefore, the best transcription termination site capable of increasing the expression vector of the expression vector is SPA-GTF (gastrin termination site of poly-A of SV40) is most preferred. Accordingly, the present invention can increase the expression titer of a foreign protein by using the transcription terminator combination of the group as a transcription termination region of an existing expression vector, and provides an expression vector pSG in the present invention.

The pSG vector introduces SPA-GTF at the transcription termination site of the pSV vector and is a vector composed of 3309 bp. The base sequence of the vector was prepared as SEQ ID NO: 7 and attached. As a result of introducing β-gal into the pSG vector of the present invention, the expression titer was found to be four times higher than that of the conventional pSV vector, which is the nucleic acid base derived from the transcription termination region of the gastrin gene. It can be seen as a result of reducing the instability of the mRNA transcript, including the transcription termination site through the combination of the nucleic acid base derived from poly-A of the SV40 virus.

Therefore, the vector combining the transcription termination site with SPA-GTF in the conventional vector of the present invention can be used to obtain a useful protein such as a bioactive substance by expressing a foreign gene in an animal cell line.

In addition, the present invention provides an expression vector including both sites capable of increasing the expression potency of the foreign gene in the animal cells mentioned above. These two sites are transcription termination sites in which the beta globin MAR factor of the reverse body and the gastrin termination site are combined with poly-A of SV40.

The structure of the expression vector pMSG of the present invention is shown in FIG. 13, and a total nucleotide sequence of 6347 bp was prepared in the sequencing software of SEQ ID NO: 8 and deposited in a depository institution to receive KCCM 10202. More detailed maps of pMSG are shown in Table 1 below.

TABLE 1

SEQ ID NO: function 1-419 Early promoter and enhancer of SV40 virus 420-448 Multiple Cloning Site (MCS) 449-656 Enterprise Termination Site 3365-6329 Beta globin MAR factor reverser 1272-2132 Beta lactamase (β-lactamase: Amp ^R )

In order to analyze the efficacy and usefulness of the pMSG of the present invention, it selectively binds to TGF-β, a human cell active protein, to induce protein expression of TGF-βSRII gene, which can inhibit the side effects of TGF-β. As a result, as shown in FIG. 14, the TGF-βSRII protein expressed through the pMSG expression vector has a glycoprotein form of a typical animal cell in which a sugar chain structure of the protein is formed to increase the molecular weight of the original protein. The initial expression of TGF-βSRII was about 100 ng / 10 ⁶ cell / day, and most of the cells were expressed evenly, and as shown in FIG. 15, MTX was added to 1 μM of cells expressing TGF-βSRII protein. A maximum of 10 ug / 10 ⁶ cell / day could be obtained. The TGF-β plays various roles in vivo, and in particular, it is regarded as a factor that causes renal glomerulosclerosis, cirrhosis of the liver, keratinization of epidermal cells, inflammation of the occlusal cartilage, and the treatment of TGF-βSRII as a treatment for TGF-β. A method of suppressing the

Therefore, the system using the pMSG vector of the present invention has improved the lack of protein expression amount and difficulty in securing the expression cell line, which is a problem with the existing expression system, various kinds of physiologically active substances such as by implementing a conventional animal expression system Can produce a large amount of recombinant protein.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Preparation of pMS-β-gal Vector

(1) Preparation of pMS-β-gal Vector

To prepare the pMS-β-gal vector, several processes were performed.

① Pure separation of genomic nucleic acid from G-2 cells to secure the nucleotide sequence of beta globin MAR (human β-globin 5 'MAR)

Genome DNA was isolated from human cell line G-2 using Promega's Wizard Genomic DNA purification kit from the US. For details, follow the experimental procedure provided by the supplier.

② Genomic nucleic acid target PCR method and subcloning

In order to secure the nucleotide sequence of the beta globin MAR, the pure genomic DNA isolated above is used as a template, and the primers correspond to the BML1 corresponding to the front part (5 ') and the back part (3') of the beta globin MAR. BMR1 was used to perform a polymerase chain reaction (PCR). Primer BML1 (SEQ ID NO: 9) and Primer BMR1 (SEQ ID NO: 10) used. The polymerase chain reaction consisted of 32 cycles. Each cycle is shown in Table 2 below.

TABLE 2

Condition Cycles One 94 ℃, 2 minutes One 2 94 ℃, 30sec-> 60 ℃, 45sec-> 72 ℃, 45sec 2-31 3 72 ° C., 10 minutes 32

Subcloning was completed by inserting the product obtained by PCR into the pT7blue vector of Novagen (Novagen) to prepare a pT7blue / betaglobin MAR vector. Novagen's pT7blue vector is a TA cloning vector designed to directly clone the product of a polymerase chain reaction.

③ Recombinant vectors (version I and version II vectors) incorporating a restriction site (MCS: multicloning site) useful in vitro PCR mutagenesis (5 ') in front of the pSV-β-gal vector promoter. Produce

In order to efficiently clone a number of MAR factors including the beta globin MAR factor subcloned into the pT7blue vector to the promoter of the pSV-β-gal vector, the recombinant pSV-β-gal version I of FIG. Version II vectors were constructed.

In order to construct a recombinant pSV-β-gal version I vector, a 443bp Spe I--Hind III fragment containing an SV40 promoter was processed by treating the pSV-β-gal vector with restriction enzymes Spe I and Hind III. After recovery from the BIO 101's gene clean III kit and recovery, the fragments were inserted into the pBluescript SK (+) (Stratagene, USA) vector, which was subjected to the same restriction enzymes Spe I and Hind III. First, a pBluescript / SV40 I promoter vector was constructed. Next, by treating the pBluescript / SV40 I promoter vector prepared above with restriction enzymes Sca I and Hind III, the fragment containing the SV40 promoter was separated and recovered from agarose gal in the same manner as described above, and then treated with the same restriction enzyme. Version I vector was completed by inserting into the ring-opened pSV-β-gal vector.

In addition, in order to construct a recombinant pSV-β-gal version II vector, EcoR I and Hind III were treated with pSV-β-gal vector to isolate and purify the 420 bp EcoR I / Hind III fragment containing the SV40 promoter by the above method. After recovery, the fragment was treated with the same restriction enzymes EcoR I and Hind III and inserted into the ring-opened pBluescript SK (+) vector to prepare a pBluescript / SV40 II promoter vector. Next, by treating the pBluescript / SV40 II promoter vector prepared above with restriction enzymes Sca I and Hind III, the fragment containing the SV40 promoter was separated and recovered from agarose gal in the same manner as described above, and then the same restriction enzyme was obtained. The version II vector was completed by inserting and linking to the ring-opened pSV-β-gal vector.

④ pMS-β-gal vector preparation by inserting beta globin MAR factor into the back of the pSV-β-gal vector promoter (5 ')

After treating the pT7blue / betaglobin MAR vector prepared in ② with restriction enzymes Spe I and Sma I, a 3 kb sized DNA fragment containing beta globin MAR factor was isolated and purified from agarose gal, and recovered. Phosphorus Spe I and Sma I were treated and inserted into the ring-opened recombinant pSV-β-gal version I vector to complete beta globin MAR factor cloning. To determine the orientation of the inserted beta globin MAR factor, the restriction enzyme Hind III present in the restriction enzyme beta globin MAR factor and present in the recombinant pSV-β-gal version I vector was confirmed to be reverse.

(2) Determination of expression titer of pMS-β-gal vector

Simultaneous transduction of 2 ug of a plurality of test vectors containing the prepared pMS-β-gal vector together with pSV2Neo vector, a selection vector, using DOSPER (Boehringer Manheim, Germany), a kind of fat surfactant, in CHO DG44 cells (co-transfection) method was introduced, and as a control, pSV-β-gal vector was also performed. CHO DG44 cells incorporating vector DNA were placed in screening medium containing G418 (neomycin) (MEM-α medium: nucleoside containing 10% heat treated FBS, 850 ug / ml G418). After incubation, 20 positive cell lines resistant to G418 were isolated. The isolated 40 positive cell populations were analyzed by the quantitative Southern blotting to analyze the number of β-gal genes and the selection of neo genes, which are selection marker genes, and Northern blots to control vector pSV-. The amount of β-gal RNA transcribed in β-gal and pMS-β-gal vectors was analyzed.

FIG. 6 shows the number of β-gal genes (control: a, the present invention: b) and the number of transcribed RNAs in animal cells including the control vector pSV-β-gal or pMS-β-gal vector of the present invention. c, the present invention: d), and the selectable neo gene number (control: e, the present invention: f) was analyzed by Southern blot and Northern blot, Figure 7 is a control vector pSV-β-gal or pMS-β The graph shows the correlation between the number of β-gal genes inserted into animal cell lines containing -gal vectors and the amount of β-gal expression. The control vector had a very small amount of RNA transcribed in DNA relative to the number of inserted genes of a as shown in c. However, in the pMS-β-gal vector of the present invention, the number of RNAs transcribed from the inserted gene was as high as d. In addition, in FIG. 7 comparing the number of genes and the amount of gene expression, the control cell line (O) exhibits a titer of 10 times or less than the pMS-β-gal vector of the present invention because β-gal activity is independent of the number of genes. In the cell line (●) to which the pMS-β-gal vector was introduced, the number of genes was 10 or more (copy) on average, and the amount of protein expression was increased even in cell lines with a large number of genes.

(4) Expression pattern of pMS-β-gal vector according to cell line.

In addition to CHO cells, several kinds of cells are used to express foreign genes through animal cells, and the expression amount of foreign genes varies depending on each cell. Therefore, in the present invention, since the above expression system confirmed the fact that the expression titer and the expression frequency of the expression cell line were increased in the CHO cell line, the vector of the present invention was applied to animal cells having different origins and forms of cells from the CHO cell line. Was tested for applicability.

The pSV-β-gal vector and pMS-β-gal vector used as a control group were transferred to BHK (Baby Hamster Kidney cells), NIH3T3 (Mouse fibroblast cells), and HEK293 (Human Embryonic Kidney cells), respectively. Experimental method of the above (3) to increase the production frequency of the positive cell line and the amount of β-gal expression in the positive cell line in cultured cells containing G418 and then cultured for about 14 days in G418 containing medium. Investigation was carried out in the same manner as

8 shows the expression titers of the pMS-β-gal vector of the present invention in various cell lines. The BHK cell line shows a similar pattern to that of the CHO cell line. No difference from the pSV-β-gal expression vector was observed. The same effect was measured in the production frequency of positive cell lines. Therefore, the effect according to specific cells of the expression vector of the present invention is changed depending on the target cells and it is difficult to expect the equivalent effect in all animal cells, but useful in CHO, BHK, and NIH3T3 cell lines widely used for gene expression of animal cells. Can be used.

(5) Establishment of expression system using pMS-β-gal vector

In the existing expression vector system, long-term cell culture and superior cell lines showing the maximum expression level from a plurality of candidate cell lines are required to be selected. In order to exclude the inefficiency according to the selection of the superior cell line and to express the foreign gene to the maximum, a method of establishing the maximum expression cell line using MTX (Metho trexate) as a cell selection agent was applied.

After the pMS-β-gal vector of the present invention was introduced into cells, changes in protein expression according to the addition of MTX were investigated. The pMS-β-gal vector and the control vector (pSV-β-gal) were introduced into CHO DG44 cells lacking the DHFR gene together with the pDCH1P vector having the DHFR gene by co-transfection. The cell lines into which the DHFR gene was introduced were cultured in selection medium (nucleoside-free MEM-α medium containing 10% of heat treated and dialyzed FBS), and then a large number of β-gal expression positive cell lines were selected by β-gal staining. . β-gal staining for selection of β-gal expressing positive cells was first fixed in cells grown in selection medium by treatment with 2% formaldehyde and 0.2% glutaraldehyde at 4 ° C. for 10 minutes. After washing twice with PBS as a washing solution, X-gal, a substrate of β-gal enzyme, is treated and stained using an enzyme-substrate reaction. As a result of staining, when β-gal is expressed, X-gal is decomposed to produce a blue colored reaction product, which eventually stains the cells blue. Multiple individual cell lines selected by β-gal staining were selected to sequentially increase MTX (methotrexate) to 10 nM, 20 nM, 50 nM, 100 nM, 400 nM, and finally 1 uM concentration in cell culture selection medium. Cultured in the medium was carried out gene amplification process. The previously selected cell lines were incubated at one concentration of MTX concentration for about 2-3 weeks until they adapted to each MTX concentration and grew healthy. During gene amplification, the expression level of the corresponding foreign gene, β-gal, was measured using conventional ELISA and β-gal activity assay.

Figure 9 shows the change in the expression level of foreign protein according to the amount of MTX addition in the cells introduced pMS-β-gal vector and the control vector of the present invention. In general, induction of maximal expression of foreign genes in CHO cell lines results in a rate of production (approximately 10 μg / 10 ⁶ cell / day) of approximately 2.5% of the total protein (Kaufman, 1997. Methods Mol Biol 62). , 287-300). In the present invention, the number of gene amplifications for a cell line adapted to a concentration of 1000 nM by gradually increasing the amount of MTX was found to be about 100 to 1000 genes amplified. As a result of investigating the expression levels of pMS-β-gal expression vector and control protein (pSV-β-gal) in these gene-amplified cell lines, the expression vector was significantly different between cell lines. On the basis of 20 μg / 10 ⁶ cells, which is a level of useful protein production, it was confirmed that the control group appeared at a frequency of 25% while the expression vector in the present invention appeared at a frequency of 88%. In addition, the pMS-β-gal expression vector of the present invention was found to be excellent even when comparing the cell lines showing the maximum expression level, so that a large amount of protein can be induced by inserting a foreign gene into pMS (KCCM-10203). The above phenomena can be expected to yield higher yields and efficiencies than conventional expression vectors in order to establish a high level of protein-producing cell lines in the future when using the expression vector of the present invention to express useful foreign genes.

Example 2 pSPUK, pMSPUK, pCPUK, pMCPUK vector production

(1) pCMV vector production

In order to facilitate cloning of the scu-PA gene into the CMV promoter and SV40 promoter expression vectors, a pCMV vector was first constructed. To prepare the pCMV vector, polymerase chain reaction (PCR) was carried out on the pcCDNA 3.1 (+) (Invitrogen, USA) vector, including the CMV promoter portion, the multiple cloning site (MCS) portion, and the transcription termination region. It was. The front (5 ') primer of PCR is CMVL1 of SEQ ID NO: 11, and the back (3') primer is PAR1 of SEQ ID NO: 12. The CMVL1 primers had restriction enzymes SacII, Cla I, Nru I sites, and the PAR1 primers were prepared so that the Bsm I sites were inserted to facilitate cloning. The PCR products of about 1.4 kb obtained using these primers were digested with Sac II and Bsm I, and then inserted into the recombinant pSV-β-gal version I vector which had been previously treated with Sac II and Bsm I to bloom. pCMV vector was completed.

(2) Securing the scu-PA gene and constructing expression vectors pSPUK, pMSPUK, pCPUK and pMCPUK expressing scu-PA

To prepare recombinant vectors for scu-PA expression in animal cells, genomic DNA is isolated and isolated from recombinant CHO cell lines with scu-PA genes derived from human TCL-598 cell lines but with low expression. The enzyme chain reaction to secure the scu-PA gene. The sense primer (5 ') PKL1 used for PCR is SEQ ID NO: 13, and the base sequence of the antisense primer (3') PKR1 is SEQ ID NO: 14. PKL1 primers were prepared with Hind III restriction enzyme sites and PKR1 primers with Sma I restriction enzyme sites inserted therein to facilitate cloning.

About 1.3 kb of the scu-PA PCR product (a DNA fragment containing the base of -6 of the human scu-PA gene to the base of 1293 of the coding region) obtained using these primers was sequentially converted into Sma I and Hind III restriction enzymes. After cleavage, the pCPUK expression vector was prepared by inserting the plasmid pCMV vector previously digested with restriction enzymes Hind III and EcoR V.

In addition, the recombinant pSV-β-gal version I vector prepared above was treated with Sma I and Hind III to isolate and purify fragments containing the SV40 promoter, and then inserted into a control pCPUK previously opened with restriction enzymes Nru I and Hind III. In conjunction, pSPUK was completed.

In order to insert the MAR factor into the pCPUK vector, the pCPUK vector was subjected to ring opening by treating the restriction enzymes Sac II and Nru I and then isolated by treating the pMS-β-gal vector prepared above with the restriction enzymes Sma I and SacII. The pMCPUK vector was completed by inserting and connecting the globin MAR factor.

In order to insert the beta globin MAR factor into the pSPUK vector, the pSPUK vector was opened by treatment with restriction enzymes Stu I and Sac II, and then isolated by treating the pMS-β-gal vector with restriction enzymes Stu I and Sac II. The pMSPUK vector was completed by inserting and connecting the factors.

Therefore, pSPUK having scu-PA inserted in the existing vector pSV was prepared, and pMSPUK was prepared by inserting a reverse MAR factor into the pSPUK. In addition, pCPUK having scu-PA inserted into the existing vector pCMVdp was prepared, and pMCPUK was prepared by inserting a reverse MAR factor into the pCPUK.

(3) measuring the efficiency of pSPUK, pMSPUK, pCPUK and pMCPUK vectors

① Intracellular Introduction of Recombinant Scu-PA Gene, Recombinant Cell Line Selection and Gene Amplification

Four recombinant expression plasmids, pSPUK, pMSPUK, pCPUK and pMCPUK, were introduced into DHFR ^- CHO cells (CHO DG44), respectively, to obtain recombinant stable cell lines expressing large amounts of recombinant scu-PA. 2 x 10 ⁵ CHO cells were placed in a 6-well culture vessel and incubated in a 5% CO ₂ reactor at 37 ° C. for 24 hours, followed by 1 ug of pSPUK, pMSPUK, pCPUK and pMCPUK expression plasmids and 10 ng of DHFR minigene. After mixing at a ratio of 1: 1, lipids such as lipofectamine (GibcoBRL) or Dosper (Loche) were introduced into CHO cells. About 6 hours after introduction, the culture medium was changed to growth medium and cultured again for 48 hours. The grown cells were subcultured using selective medium at a ratio of 10: 1, and cultured for about 2 weeks or more while changing the medium every 4-5 days. The formed cell colonies were each independently isolated or pooled together and cultured.

② Determination of scu-PA Activity in Cell Cultures

The scu-PA activity secreted in the cell culture medium was shown by comparing the urokinase activity with the standard amidolytic activity based on the S-2444 peptide of the culture medium. First, 1 x 10 ⁶ cells were placed in a 6 well culture vessel together with 2 ml of growth medium and incubated for 17 hours at 37 ℃, 5% CO ₂ incubator. In order to measure the amidolytic activity of the obtained culture supernatant, 50 μl of serially diluted culture supernatant was placed in a 96 well plate and 30 μl assay buffer solution (50 mM Tris / HCl (pH 8.8), 80 mM NaCl, 0.02% Tween80). Recombinant scu-PA was then activated by adding 10 μl of plasmin (0.5 U / ml) and reacting at 37 ° C. for 20 minutes. 10 μl of aprotinin (100KIU / ml) was added to inhibit plasmin activity and 100 μl of chromogenic substrate solution (containing 6 mM S-2444 in the assay buffer solution) was mixed with the reaction solution. After reacting at 37 ℃ for 1 hour. Scu-PA activity and concentration in the culture were determined by measuring the 405 nm absorbance of the final reaction solution with a microplate reader and comparing urokinase absorbances as standard.

Figure 10 shows the expression titers of scu-PA by introducing the pMSPUK, pMCPUK, pSPUK and pMCPUK vectors of the present invention into the CHO cell line, pMS, pMC vector gene expression is about 4 than pSV, pCMV expression vectors of control vectors The increase was more than doubled.

Example 3 Preparation of pSG-β-gal Vector

(1) Securing transcription termination site of gastrin gene and preparing pSG-β-gal vector

In the transcription termination region of the gastrin gene, the sense strand was synthesized by SEQ ID NO: 15, and the antisense strand was synthesized by SEQ ID NO: 16 to be annealed with each other. The binding reaction product was treated with the restriction enzymes BamH I and Pst I, and then inserted into the pSV-β-gal vector (Promega, USA), which had been previously treated with the same restriction enzyme to complete the pSG-β-gal vector. .

(2) measuring the efficiency of the pSG vector

Co-transfection of the pSG-β-gal vector prepared in (1) with the pSV2Neo vector, a selection vector, using DOSPER (Boehringer Manheim, Germany), a kind of fatty surfactant in Cos-7 cell line (co-transfection) The expression titers of β-galactosidse were measured using the method.

Figure 12 shows the expression titers of β-gal of the pSG-β-gal vector, the combination of pSG expression vector, that is, the combination of the SV40 poly-A signal and the gastrin transcription terminator among the combination of proteins compared to the conventional SV40 poly-A signal The effect of increasing the expression amount of about 4 times was obtained. Therefore, it is possible to produce a large amount of protein by introducing a foreign gene into the pSG-β-gal vector.

Example 4

(1) pMSG vector production

A vector to which beta globin MAR reverse body and SPA-GTF were simultaneously applied was prepared by a polymerase chain reaction method as shown in FIG. 13. In order to prepare pMSG vectors, three sets of polymerase chain reactions were carried out using pMS-β-gal and pSG-β-gal as templates, and then a specific restriction enzyme was connected to the reaction product to complete the pMSG vector.

① Polymerase chain reaction of beta globin MAR factor

PCR was performed using the used sense primer ML1 (SEQ ID NO: 17) and the antisense primer MR1 (SEQ ID NO: 18). The obtained reaction product was digested with the restriction enzymes Sac II and Cla I, and then subjected to the same restriction enzyme and inserted into the ring-opened pMS-β-gal vector to complete the one-step subcloning to obtain the intermediate vector product pMS-. β-gal / sc vector was constructed.

② polymerase chain reaction including multiple cloning site and transcription termination site

The gastrin transcription termination site of the pSG vector was PCR using senseprimer TL1 (SEQ ID NO: 19) and antisense primer TR1 (SEQ ID NO: 20). By subcloning the pGEM-T Easy vector (Promega, USA), a TA cloning vector designed to directly clone the reaction product without restriction enzyme treatment, pGEM-T / MCSp (A ) Was produced.

③ Polymerase chain reaction of SV40 promoter and multiple cloning site

Using the sense primers PL1 (SEQ ID NO: 21) and PR1 (SEQ ID NO: 22), the SV40 promoter and the multiple cloning site moiety were PCR in version I of FIG. The reaction product was treated with the restriction enzymes Apa I and Bgl II, and then linked to the vector pGEM-T / MCSp (A) vector, which was previously ring-opened by treating the same restriction enzyme, to the pGEM-T / SVMCSp (A) vector. Was prepared as an intermediate.

④ Construction of pMS vector and pMSG vector

PGEM-T / SVMCSp (A) vector prepared in ③ of Example (4) was treated with restriction enzymes Apa Ia I and BamH I to isolate and purify DNA fragments composed of SV40 promoter, multiple cloning site and SV40 transcription termination site. Subsequently, the same restriction enzyme was treated beforehand and inserted into the pMS-β-gal / sc vector prepared in ① of Example (4), thereby completing a pMS vector. To prepare the pMSG vector, the pSG vector prepared above was treated with restriction enzymes BamH I and Sca I to isolate and purify a DNA fragment of 950bp having a GTF sequence of gastrin, and then the ring-opened pMS was previously removed by the same restriction enzyme. Linkage insertion into the vector completes the pMSG vector.

(2) Determination of expression titers of pMSG expression vectors

In order to verify the efficacy of the foreign gene expression in the CHO cell line of pMSG prepared in Example 3 (1) and to investigate the industrial applicability, the gene of the glycoprotein TGF-βSRII (TGF-βsoluble receptor II) is sense primer PCR was performed using TRI (SEQ ID NO: 23) and antisense primer TRR1 (SEQ ID NO: 24). The amplified TGF-βSRII gene was cloned into the pMSG vector and the control vector pSV on the Nhe I and Xho I restriction enzymes. Introduced. After culturing for about 2 weeks in a selection medium capable of growing only the cell line containing the DHFR gene, a large number of cell lines were obtained, and the amount and characteristics of TGF-βSRII expressed from these cell lines and secreted out of the cell were examined by immunoblot method. .

Figure 14 shows the expression pattern of TGF-βSRII of the pMSG vector of the present invention by antigen antibody response, a is the expression pattern of TGF-βSRII in the control vector pSV, b is the expression pattern of TGF-βSRII of the pMSG vector. In the control group, only one cell line secreted TGF-βSRII in 25 growth cell lines, whereas 23 cells in 25 growth cell lines expressed TGF-βSRII using the pMSG expression vector. In addition, the characteristics of TGF-βSRII protein expressed through pMSG expression vector showed that the sugar chain structure of the protein was formed to increase the molecular weight than the size of the original protein, which was a typical animal cell glycoprotein form. Most cell lines showed an expression level of about 100 ng / 10 ⁶ cell / day evenly.

In order to maximize TGF-βSRII expression of a cell line transformed with pMSG / TGF-βSRII of the present invention, amplification of the gene was induced by gradually increasing the concentration of MTX in a medium containing a MTX selector.

Figure 15 shows the expression of TGF-βSRII by cloning the TGF-βSRII gene in the pMSG vector of the present invention, transforming the CHO cell line, and adding MTX to TGF-βSRII expressing cells. In the control group, cell growth was inhibited by the increase of MTX concentration in the gene amplification process, but there was no inhibition of cell growth in the case of pMSG / TGF-SRII. This phenomenon is believed to be due to the result that the MAR factor of the pMSG expression vector of the present invention increases not only the foreign gene but also the expression of the DHFR gene used for selection. As shown in FIG. 15, when the amplification of the gene was induced by increasing the amount of MTX in the medium to 1 uM in a positive cell line, the maximum expression amount of TGF-βSRII was about 10 μg / 10 ⁶ cell / day A large number of cell lines expressing could be obtained.

TGF-β plays various roles in vivo, and it is considered as a factor that causes renal glomerulosclerosis, cirrhosis of the liver, keratinization of epidermal cells, inflammation of occlusal cartilage, and excessive TGF-βSRII when treated with TGF-βSRII. Inhibition of -β may produce a therapeutic effect. In developing such therapeutic agents, TGF-β SRII from the CHO cell line made in the present invention may exhibit a superior therapeutic effect than proteins expressed from lower cells such as E. coli or Pichia pastoris, and the expression titers of foreign proteins in animal cells. Increased expression vectors can be usefully used.

As mentioned above, the present invention improves the expression suppression caused by the surrounding base of the host cell, which is a problem that frequently occurs when foreign genes are expressed in animal cell lines, and induces an increase in the number of foreign genes. An expression vector pMS (KCCM-10203) vector was prepared, which can exhibit a synergistic effect of gene expression about 8 times than that of gene expression.

In addition, the present invention is an expression vector pSG having a transcript termination region which can induce transcription termination at a specific region of the gene transcript of an animal cell and can express about 3 times as much expression protein as the existing gene transcript polyA region. Vectors were prepared.

The present invention completed a pMSG (KCCM-10202) expression vector by combining a nucleic acid region having the above function and a multiple cloning site to which foreign genes can be applied in the future. 10 ug / 106 cell / day production rate was confirmed. Therefore, the expression vector of the present invention can be used to effectively express the expression of foreign genes by using animal cell expression vectors and can be produced in animal cells as recombinant proteins having the same structure and function of higher animal proteins expressed in lower animals. Do.

Claims (8)

An animal cell expression vector comprising a reverse matrix of the MAR factor of the beta globin gene or the MAR factor of the beta globin gene at the 5 'end of a conventional vector promoter. The animal cell expression vector of claim 1, wherein the animal cell expression vector is pMS KCCM 10203. The animal cell expression vector of claim 1, wherein the animal cell expression vector is pMC. An animal cell expression vector comprising a combination of a transcription termination site of a gastrin gene in a poly-A (polyadenylation) signal of a SV40 virus of SEQ ID NO: 3 as a transcription terminator in a conventional vector. The animal cell expression vector of claim 4, wherein the animal cell expression vector is pSG. PMGS KCCM 10202 vector of SEQ ID NO: 8 comprising a Nuclear matrix attachment region element reverse of the beta globin gene and a transcription terminator combination of poly-A and gastrin gene of SV40 virus. Method for producing a bioactive material in animal cells using a vector selected from the group consisting of the expression vector of claim 1, the expression vector of claim 4 and the expression vector of claim 6. The method of claim 7, wherein TGF-βSRII is prepared using the expression vector of claim 6.